Effect of the inhibitor-resistant M69V substitution on the structures and populations of trans-enamine beta-lactamase intermediates.

Abstract

The objective of this study was to determine the molecular factors that lead to beta-lactamase inhibitor resistance for the M69V variant in SHV-1 beta-lactamase. With mechanism-based inhibitors, the beta-lactamase forms an acyl-enzyme intermediate that consists of a trans-enamine derivative in the active site. This study focuses on these intermediates by introducing the E166A mutation that greatly retards deacylation. Thus, by comparing the properties of the E166A and M69V/E166A forms, we can explore the consequences of the resistance mutation at the level of the enamine acyl-enzyme forms. The reactions between the beta-lactamase and the inhibitors tazobactam, sulbactam, and clavulanic acid are followed in single crystals of the enzymes by using a Raman microscope. The resulting Raman difference spectroscopic data provide detailed information about conformational events involving the enamine species as well as an estimate of their populations. The Raman difference spectra for each of the inhibitors in the E166A and M69V/E166A variants are very similar. In particular, detailed analysis of the main enamine Raman vibration near 1595 cm(-1) reveals that the structure and flexibility of the enamine fragments are essentially identical for each of the three inhibitors in E166A and in the M69V/E166A double mutant. This finding is in accord with the X-ray-derived structures, presented herein at 1.6-1.75 A resolution, of the trans-enamine intermediates formed by the three inhibitors in M69V/E166A. However, a comparison of Raman results for M69V/E166A and E166A shows that the M69V mutation results in a 40%, 25%, and negligible reductions in the enamine population when the beta-lactamase crystals are soaked in 5 mM tazobactam, clavulanic acid, and sulbactam solutions, respectively. The levels of enamine from tazobactam and clavulanic acid can be increased by increasing the concentrations of inhibitor in the mother liquor. Thus, the sensitivity of population levels to the inhibitor concentration in the mother liquor focuses attention on the properties of the encounter complex preceding acylation. It is proposed that for small ligands, such as tazobactam, sulbactam, and clavulanic acid, the positioning of the lactam ring in the active site in the correct orientation for acylation is only one of a number of poorly defined conformations. For tazobactam and clavulanic acid, the correctly oriented encounter complex is even less likely in the M69V variant, leading to a reduction in the level of inhibition of the enzyme via formation of the acyl-enzyme intermediate and the onset of resistance. Analysis of the X-ray structures of the three intermediates in M69V/E166A demonstrates that, compared to the structures for the E166A form, the oxyanion hole becomes smaller, providing one explanation for why acylation may be less efficient following the M69V substitution.

Electron density of the inhibitors in the active site of M69V/E166A SHV-1 β-lactamase. The omit Fobs− Fcalc difference electron density of the active site of the double mutant protein shows the density of covalently bound trans-enamine intermediate for tazobactam (A), sulbactam (B), and clavulanic acid (C). The omit density is contoured at 3.0 σ (red), 2.0 σ (blue), and 1.5σ (green) and is calculated at the end of refinement after omitting the ligands from the map calculation. The omit map suggested clavulanic acid is in a decarboxylated state similar to that observed when complexed in the E166A single mutant structure.

Stereo diagrams depicting the active sites of the inhibitor bound structures of the E166A and inhibitors resistant M69V/E166A variants. For each of the inhibitors, the M69V/E166A double mutant (grey with colored N, O, and S atoms) and the single E166A mutant (black lines) are superimposed. Tazobactam (tazo, 3A), sulbactam (sulb, 3B), and clavulanic acid (clav, 3C) are shown covalently attached to S70. The E166A SHV-1 β-lactamase structures used for the super positions have PDB identifiers 1RCJ (complexed with tazobactam), 2A49 (complexed with sulbactam), and 2A3U (complexed with clavulanic acid) with E166A) are used for comparison of the respective inhibitor bound double mutant structures. Water molecules present near the inhibitor are shown as small spheres. The catalytic water involved in deacylation (W1) is highlighted. For tazobactam in the single mutant structure, a ligand induced shift causes N170 to reorient thereby shifting the position of catalytic water to a new position (W2). Two additional waters are depicted in the tazobactam bound structures (W3 & W4) which are key for interacting with either tazobactam and K234 and/or R244.

Time dependence of the enamine peak area near 1593 cm−1 (normalized to the amide I band) for the E166A (upper trace) and M69V-E166A (lower trace) SHV β-lactamase variant crystals and tazobactam (5mM in the mother liquor)

Time dependence of the enamine peak area near 1610 cm−1 (normalized to the amide I band) for the E166A (upper trace) and M69V-E166A (lower trace) SHV β-lactamase variant crystals and clavulanic acid (5mM in the mother liquor)

Time dependence of the enamine peak area near 1599 cm−1 (normalized to the amide I band) for the E166A (cyan squares) and M69V-E166A (blue diamonds) SHV β-lactamase variant crystals and sulbactam 5mM in the mother liquor

Crystallographically observed shifts in the vicinity of the M69V mutation. A) the |Fobs, M69V/E166A|-|Fobs, E166A | electron density map is contoured at 3.5 σ (green) and −3.5 σ (red). The direction of atomic shifts is indicated by a red arrow. In addition to the M69V mutation, the shifts in the strand 243–246 is readily observed in the |Fo|-|Fo| map via the positive and negative shifts peaks (phases obtained from the M69V model). The latter strand movement is likely a result of the energetic need to partially fill up the void generated by the M69V mutation. In addition, residue N170 shifted as well to accommodate the branced valine side chain. B) the |Fobs, M69V/E166A|-|Fobs, E166A | electron density map near the oxy-anion hole and acyl-bond is contoured at 3.0 σ and −3.0 σ. Positive and negative shift peaks in the protein are colored green and red, whereas positive and negative shift peaks in tazobactam are colored purple and blue, respectively.